Rescuing plasmid from dead E.coli stock.

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Fri Dec 6 05:04:00 EST 1996


In article
<Pine.SGI.3.91.961205095147.17816B-100000 at hancock.microbiology.ubc.ca>,
emma at HANCOCK.MICROBIOLOGY.UBC.CA (Emma Macfarlane) wrote:

> I remember reading some time ago on this newsgroup about retreiving 
> plasmid from dead plates to re-transform into E.coli, but I can't
> find reference to it in the archives. Is it possible to
> do the same thing with frozen stocks? We have an important construct 
> in a sickly E.coli strain that is now refusing to grow. I tried
> taking a scraping of cells, boiling it up in water, spinning it down
> and using the supernatant to transform DH5a. This didn't work.
> If anyone has an alternative suggestion I would appreciate hearing it.
> Please reply direct to my email address emma at hancock.microbiology.ubc.ca 
> as I am not a regular subscriber to the newsgroup.
> Thanks in advance
> Emma


I recommend you do a normal miniprep (Birnboim) of a small aliquot of your
old stock and use some of the DNA for a new transformation. I have done
that successfully several times, with good success. Normally I take 1ul of
50ul from the miniprep for a transformation (dont run it on a gel, you
wont see anything as the yield is very low). This is usually enough to get
a lot of clones, even when you start from only 10ul of glycerol stock. You
can even do the recovery with dried-out agar plates: cut out a piece where
a clone was, drop the piece in GET for several hours to overnight, then do
a normal miniprep without removing the agar pieces before (looks terrible,
but works). Of course you can also use stab culture stocks to do the
recovery. 

Hope it helps,
Frank

-- 
Dr. Frank O. Fackelmayer
Division of Biology
University of Konstanz
D-78434 Konstanz
Germany



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